Placental Growth Factor (PlGF) is a member of Vascular Endothelial Growth Factor (VEGF) family and acts as angiogenic amplifier by signaling through VEGF receptor-1, expressed mainly by vascular endothelial cells, monocytes, macrophages, but also by human CD34+ marrow repopulating stem cells.
PlGF occurs in at least four splicing isoforms, PlGF-1 to PlGF-4 that differ in size and binding properties.
The complete peptide and nucleotide sequence of PlGF-1 has been described in patent application WO 92/06194.
In details, PlGF-1 sequence contains nine cysteine residues in position 35, 60, 66, 69, 70, 77, 111, 113 and 125 that are potential candidates for the formation of intra- and inter-chain disulfide bonds. The above numbering of the cysteine residues is relative to recombinant PlGF-1 produced in Escherichia coli which comprises an N-terminal Methionine not present in native PlGF-1 (for example: Cys35 in the recombinant PlGF-1 corresponds to Cys34 in native PlGF-1—also known as PlGF131).
The presence of disulfide bonds has been confirmed by the analysis of the crystal structure of PlGF-1 at 2.0 Å resolution (Iyer S. et al., J. Biol. Chem., 2001, 276, 12153-12161). According to Iyer et al., PlGF-1 is described as a homodimeric molecule organized in an antiparallel arrangement covalently linked by two interchain disulfide bonds between Cys60-Cys69 Cys69-Cys60 and three intrachain disulfide bonds: Cys35-Cys77, Cys66-Cys111 and Cys70-Cys113.
The most relevant feature evidenced by the crystallographic analysis is the presence of a cysteine-knot motif. The C-terminal sequence of PlGF-1, following the position 114, does not show an organized structure and, by consequence, the related crystal structure has not been defined.
Yet, the distance existing between the two cysteine residues in position 125 in the homodimer renders the presence of an interchain covalent bond between these two positions unlikely, since this would require a distortion of the molecule.
The homodimeric form has been shown to be the biologically active form of PlGF, while the monomer of PlGF is inactive.
A number of studies have evidenced the pharmacological activity of PlGF-1.
For example, preliminary observations have shown positive effects obtained by an adenoviral vector expressing hPLGF (human Placental Growth Factor) on early and late bone marrow hematopoiesis in 5-fluorouracil-suppressed mice and in the mobilization of stem/progenitor cells into the bloodstream.
Furthermore, PlGF-1 has been shown to induce angiogenesis in vivo and to stimulate the migration and proliferation of endothelial cells in vitro (Ziche M. et al., Lab. Invest., 1997, 76, 517-531). A beneficial effect of PlGF protein or gene therapy on ischemic conditions has been disclosed in e.g. WO-A-01/56593 and US-2007/0027100. The ischemic conditions include cerebral ischemia, acute myocardial infarction and ischemic hind limb.
Patent Application WO-A-03/066676, herein enclosed by way of reference, describes a process for the extraction and purification of recombinant PlGF-1 from genetically modified bacterial cells. The product obtained, hereinafter referred to as [DIM1/2] PlGF-1, comprises at least 98.5% of dimeric and multimeric active forms, at least 70% of dimeric form and less than 1.5% of the monomeric form, which is inactive.
According to this process, PlGF-1 is expressed as inclusion bodies in E. coli by recombinant DNA technology. Then it is extracted by solubilization of the inclusion bodies in a denaturant buffer, it is refolded in the presence of a redox system and subsequently purified by a two steps chromatography procedure (anionic exchange chromatography followed by reverse phase chromatography).
Although the PlGF-1 protein obtained with the process disclosed in WO-A-03/066676 is a highly pure active form, the present inventors have found that this product does not exhibit optimal stability in aqueous solution and sometime shows batch-to-batch heterogeneity (by Mass spectrometry, SDS-PAGE and IEF analysis).
WO-A-03/097688 discloses the preparation of a mutein of the PlGF-1 molecule, wherein the cysteine in position 125 of the PlGF-1 molecule has been replaced by a different amino acid unable to form disulphide bonds.
The above mutein shows a better stability in aqueous solution than the wild-type PlGF-1.
Scope of the present invention is to provide a new stabile form of active PLGF.